Considerable effort has been employed to engineer enzymes and other proteins to achieve higher selectively and/or specific activity (Matsumura and Ellington, J. Mol. Biol. 305:331-339, 2001; Rothman and Kirsch, J. Mol. Biol. 327:593-608, 2003; Aharoni et al., Nature Genetics, 37:73-76, 2005). Human trypsin-like serine proteases are an appealing target for engineering with the goal to tailor proteases to recognize a specific, predefined primary sequence within a target protein that is normally not recognized, resulting in specific spatial and temporal modulation of target activity. Trypsin-like serine proteases are also valuable research tools in molecular biology.
Manufacturing of trypsin-like serine proteases poses challenges due to their structural complexity related to the required appropriate disulfide bond formation and proper processing of the native globular polypeptide chain for activity. Furthermore, trypsin-like serine proteases often have a constricted recognition sequence limiting the absolute specificity that can be engineered into the molecules. (Gosalia et al., Mol. Cell. Proteomics, 4:626-36, 2005, US Pat. Appl. No. US20040072276A1). An alternative to human trypsin-like serine proteases, intracellular plant viral proteases that are easier to manufacture could be used as a starting point to develop therapeutics as well as new research tools.
Potyviruses are a class of plant viruses transmitted mainly by aphids, causing significant losses in pasture, agricultural, horticultural and ornamental crops annually. Typical representatives of potyviruses are Potato virus A (PVA), tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV). Potyvirus monopartite genome contains (+) stranded RNA, covalently linked to a viral encoded protein (VPg) at the 5′-end and polyadenylated at the 3′-end (Dougherty et al., The EMBO J. 7:1281-1287, 1988). The genome serves as an mRNA and a template for the synthesis of a complementary (−) stranded RNA by a polymerase translated from the viral genome. Upon entry into the cell, the virus RNA binds to endogenous ribosomes and the genome is translated as a single polypeptide chain. The large single polyprotein is subsequently processed into mature proteins by three virus-encoded proteases (Verchot et al., Virology, 190:298-306, 1992), the first protein (P1), the helper component (HC), and the nuclear inclusion protein (NIa) proteases. The NIa protease is responsible for the majority of the polyprotein processing, including the generation of mature RNA replication-associated proteins and capsid proteins (Verchot et al., Virology, 190:298-306, 1992).
The NIa proteases belong to the family of picornavirus 3C cysteine proteases (Parks et al., Virology, 210:194-201, 1995), that exhibit an extended P6-P1′ recognition sequence EXXYXQ*(S/G) (SEQ ID NO: 69) (Dougherty et al., Virology, 171:356-364, 1989). Although there are striking similarities in the recognition sequence for NIa proteases across the potyvirus members, each protease is highly specific for its own target sequence (Tozer et al., The FEBS J. 272:514-523, 2004). Structurally, NIa proteases appear to be related to trypsin-like serine proteases through divergent evolution involving replacement of NIa catalytic cysteine by serine in the trypsin-like proteases (Bazan and Fletterick, Proc. Natl. Acad. Sci. 85:7872-7876, 1988). NIa and trypsin-like serine proteases share a similar overall 3-dimensional protein fold as well as the spatial proximity of their respective catalytic residues. The 3C-like family of cysteine proteases offers several advantages over more complex extracellular proteases. They can be easily produced in the cytosol of bacteria, have no disulfide bonds, and have an extended substrate recognition sequence. The challenge of using the 3C-like proteases is their activity loss in non-reducing conditions due to oxidation of active site and/or surface exposed cysteines, therefore limiting their use (Higaki et al., Cold Spring Harbor Symposia on Quantitative Biology, 615-621, 1987). Therefore, the proteases require reducing agent to sustain their functional activity (Nunn et al., J. Mol. Biol. 350:145-55, 2005; Birch et al., Protein Expression and Purification 6:609-18, 1995). Thus, there is a need for engineered plant viral proteases that remain active in the absence of exogenous reducing agents.